Background and Objectives: Polycystic ovary syndrome (PCOS) is one of the most important endocrine disorders, which affects about 5-10% of women in reproductive age and is one of the main causes of infertility due to anovulation. In this study, the effect of letrozole was investigated on serum levels of testosterone and estradiol hormones and body and ovarian weight. Methods: This experimental study was carried out on 20 adult female Wistar rats (180-200 g) in 2 groups. The first group (control) received 0.2 ml of distilled water (solvent) and the second group (hyperandrogenism) received letrozole through gavage at a dose of 1 mg/kg bw for 21 days. At the end of this period, 6 mice of each group were randomly selected and sacrificed for hormonal studies. In the first step, blood samples were taken from the animals’ heart and centrifuged to prepare serum samples; also, uterine samples were prepared. The serum levels of testosterone and estradiol were measured by ELISA method. Data analysis was performed using independent t test. The significance level was considered p<0.05. Results: According to the obtained results, the concentration of testosterone, body weight, and ovarian weight significantly increased in the group receiving Letrozole compared to the control group (p<0.05.( Conclusion: The results of this study showed that letrozole as a non-steroidal aromatase inhibitor, blocks the conversion of testosterone into estradiol, and increases serum testosterone levels, also causes increase in body weight and ovarian weight.